The practical benefits of treatment of AIDS patients with highly active anti-retroviral therapy (HAART) are manifested by substantial increase of the life expectancy of HIV infected individuals. The current therapies target two retroviral enzymes reverse transcriptase and protease. More recently, viral entry inhibitor has been developed. However, problems associated with the toxicity of currently available drugs and emergence of new infections initiated with HAART-resistant viruses, highlight the need to continually improve available inhibitors and develop new drugs against as yet unexploited targets. Integrase (IN) is one of the most promising new targets, as its function is essential to HIV replication. The recent advances of diketo acid inhibitors of HIV integration into phase 1 clinical trails confirm this notion. Biochemical studies indicated that these compounds specifically bind IN:viral DNA structure and compete with target DNA binding. However, little is known regarding structural foundations that warrant formation of the ternary IN:viral DNA:DKA complex or how viral and host DNAs bind the IN multimer. The proposed research will address these important questions. In particular, we will employ novel experimental strategies to accomplish the following three specific aims. Specific Aim 1 will determine HIV-1 lntegrase:viral DNA contacts using domain selective cross-linking coupled with mass spectrometric foot-printing. This novel approach together with molecular modeling and site-directed mutagenesis studies should enable us to obtain detailed structural information as to how viral DNA binds IN multimer. Specific Aim 2 will employ protein and nucleic acid foot-printing to identify IN:host DNA contacts and to determine DNA conformation in the integration complex. The experimental results will be used to create a plausible molecular model for the functional nucleoprotein complex. Specific Aim 3 will characterize diketo acid (DKA) inhibitor binding site. Photo-affinity cross-linking coupled with mass spectrometric analysis will be utilized to identify new amino acid contacts to DKA; as well as disulfide DNA cross-linking and scintillation proximity assays will be used to dissect contributions of viral DNA end distortion for specific inhibitor binding. Taken together, we will obtain detailed structural information on biologically relevant IN:viral DNA:DKA complex. Such new and important structural data will facilitate the rational design of future generation inhibitors of potential clinical relevance.